Anti fasn

Western blot analysis was carried out as previously described [29 (link)]. Lysates were fractionated by SDS-PAGE and proteins were blotted on nitrocellulose membranes. Blots were pre-blocked in PBS containing 5% (w/v) dried no fat milk and then incubated overnight at 4 °C with antibodies to anti-FASN (Sigma-Aldrich, St. Louis, MO, USA), anti-DHCR24 (Cell Signaling Technology, Danvers, MA, USA), p27 kip1 (BD Biosciences, Franklin Lakes, NJ, USA) and Bim (Abcam, Cambridge, UK). Anti-actin (Sigma-Aldrich, St. Louis, MO, USA) or anti-β-tubulin (Abcam, Cambridge, UK) antibodies were used as control for loading. Antibody binding to blots was detected by chemo-luminescence (GE Healthcare, Chicago, IL, USA). Secondary antibodies were obtained from GE Healthcare. At least three independent experiments were performed. Band intensity quantification was performed using the ImageJ 1.47 v software.

Cells or tissues were lysed using buffer containing 100 mM Tris-Cl (pH 6.8), 4% SDS, 20% glycerol, and 200 mM β-mercaptoethanol. Protein was quantified by the BCA Protein assay (Pierce) using a Viento multi-spectrophotometer at 562 nm. Equal amounts of protein (15–30 μg) were separated by SDS-PAGE, transferred to Immobilon PVDF membranes (Millipore), and blocked with either 5% skimmed milk or rabbit serum in TBST (10 mM Tris (pH 7.5), 100 mM NaCl, and 0.1% Tween 20). Membranes were incubated overnight at 4°C with primary antibodies diluted in blocking buffer. The following primary antibodies were used: anti-FASN (Sigma-Aldrich, 1:1000), anti-CD133 (Miltenyi Biotec, 1:100), anti-FABP7 (Owada et al., 2006, 1:1000), anti-nestin (Millipore, 1:5000), anti-Sox2 (Millipore-Chemicon 1:1000), and anti- β-actin (Santa Cruz Biotechnology, 1:5000) antibody. Blots were washed and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at RT. Immunoreactive protein bands were visualized using ECL western blotting detection reagents (GE Healthcare UK Ltd, Amersham Place, England).

Proteins were transferred onto polyvinylidene fluoride membrane (Bio-Rad), blocked with 5% bovine serum albumin dissolved in 1× Tris-buffered saline containing 0.1% Tween-20. Anti-Flag (catalog number: F3165) and anti-FASN (catalog number: SAB4300700) antibodies were purchased from Sigma and used at a final concentration of 1:1000; anti-calnexin (catalog number: 2679S) and anti-Src (catalog number: 2109S) antibodies were purchased from Cell Signaling Technologies and used at a final concentration of 1:2000. Anti-rabbit secondary antibody (Thermo Fisher Scientific; catalog number: 31460) was used at a final concentration of 1:5000, and antimouse secondary antibody (Cell Signaling Technology; catalog number: 7076S) was used at a final concentration of 1:2000.